Researchers have long characterized samples, such as biological tissue samples, by measuring spectral response. Staining techniques deposit stains in specific locations and spatial images of the sample can often identify the spatial location of the stains and their staining targets. For example, methods such as histochemistry, immunohistochemistry, and in-situ hybridization specifically target macromolecules, nucleic acid sequences or specific antigens. After binding to such targets, these techniques deposit stains in the vicinity of the targets. In many applications, multiple staining protocols are used simultaneously and measuring the concentration of each stain in the sample provides highly relevant multiplexed data about the sample.
Often the stains overlap and the resulting spectral response of the sample represents the overlapping spectral responses of the mixture of the stains. Quantifying the separate concentrations of these coexistent stains is known as spectral unmixing. In some cases spectral unmixing can be challenging. For example, in transmission spectroscopy Beer's law governs the relation between stain concentration and transmitted light intensity and as this relation is exponential it can prove to be challenging to linear unmixing methods.
Typical spectral unmixing methods acquire an entire image cube of the sample.
Intensity data is recorded for each 2D spatial pixel at each of a number of wavelengths. Using this data, the methods determine the multiple stain concentrations present in the sample. Typically such methods collect large amounts of data for each sample and use a spectral imaging system capable of measuring sample response at each of the different wavelengths.